Obtain soil and compost samples from different environments. We have access to soil and compost samples from Christopher Hopkins from efforts to learn about the composting process of “swine lagoon” sludge and have dried and “tar-like” samples. Your instructors (Horton and Phillips) also prepared solid metagenomic DNA samples from around the campus for comparison.

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Weigh 250mg into QIAGEN PowerSoil Pro bead beating tubes.

Extract DNA following the QIAGEN PowerSoil Pro protocol in triplicate.

Elute in 50µL 5 of CD6 Elution buffer.

Quantify DNA by NanoDrop and use the Qubit BR DNA kit.

Run 1µL of sample on Agilent TapeStation using the Genomic DNA tape and reagents.

Obtain 0.8micromolar (µM) Forward and 0.8micromolar (µM) Reverse Primer for the blaCTX-M-1 antimicrobial resistance gene.

Obtain probe 0.4micromolar (µM) and dilute it with 625µL of TE Buffer.

Transfer 100µL 1 of diluted probe into tubes via pipette in a biosafety hood.

Transfer 400µL of buffer solution with 40µL of both the reverse and forward primers, and 20µL of the probe to make the primer probe mix

The primer probe mix, 3µL of the master mix, and 0.15µL of the restriction EcoRI enzyme per reaction were dispensed into the PCR plate. Then, the extracted compost DNA was added to each well that contained the overall reaction mix (1µL per reaction)

Transfer mixtures from the PCR plate to the QIAGEN Nanoplate (96 wells, 8.5k partitions) using the epMotion 5075.

Seal the Nanoplate properly with the provided seal and use roller to ensure proper seal.

Transfer the plate to the QIAcuity instrument, remove the white plate tray, and load into the digital PCR using a template for a full plate with a FAM-based probe.

Table 1. Concentrations of stocks and working solutions for primer mix and probes.

A	B	C	D
Component	Initial Concentration	Final Concentration	Volume for 500 µl (in TE)
Forward Primer	100 µM	8 µM	40 µl
Reverse Primer	100 µM	8 µM	40 µl
Probe	100 µM	4 µM	20 µl
TE Buffer	-	-	400 µl

Table 2. List of reagents used for digital PCR on the QIAGEN QIAcuity instrument.

A	B	C	D
Component and Concentration	Volume per Reaction	Final Concentration in µM	Volume for 100 reactions in 96-well plate
4X QIAcuity Probe PCR Master Mix	3 µl	1X	300 µl Master Mix
10X Primer Probe Mix (FAM channel)	1.2 µl	0.8 µM forward	120 µl Primer Probe Mix
Restriction EcoRI Enzyme 0.25 U/µl EcoRI-HF®, NEB®	0.15µl	0.8 µM reverse	15 µl NEB EcoRI-HF
RNase-free Water	6.65µl	primer	665 µl water
Template DNA	1.0 µl	0.4 µM probe	100 µl of DNA
Total Reaction Volume	12.0 µl	0.25 U/µl	1,200 µl

Table 3. Thermocycling Conditions.

A	B	C	D
Step	Time	Temperature (°C)	Number of Cycles
PCR initial heat activation	2 min	95°C	1 cycle (to activate the enzyme)
Denaturation	15 seconds	95°C	40 cycles
Combined annealing/ extension	30 seconds	60°C	40 cycles

Source: QIAGEN QIAcuity Probe Mix

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